PROJECT SUMMARY All positive-stranded RNA viruses manipulate host cell membranes for the biogenesis of their replication organelles. These structures are thought to concentrate cellular and viral factors that are required for replication and create an environment that helps to protect the virus from innate immune sensors. Enteroviruses, such as coxsackievirus B3 (CVB), and flaviviruses, such as dengue virus (DENV), represent two families of positive-stranded RNA viruses that induce the formation of distinct classes of replication organelles. Enterovirus infection results in the formation of cytoplasmic single- and double-membrane vesicles that contain endoplasmic reticulum (ER) and autophagic markers. Enteroviral replication proteins are anchored on the outer membrane leaflets of these vesicles, where they coordinate viral genome replication. In addition to infecting humans, most flaviviruses must efficiently replicate in insect vectors, such as mosquitoes and ticks. Flaviviruses induce the formation of vesicular invaginations of the ER that contain a small opening to the cytoplasm. Genome replication is mediated within these structures by the viral nonstructural proteins, which are concentrated in the vesicles. Despite the evidence that exists for the involvement of the ER in establishing these distinct replication organelles there is a lack of knowledge regarding the role of ER-associated membrane shaping regulators during enterovirus and flavivirus replication. The ER is a large, dynamic organelle that exists as perinuclear sheets and a cytoplasmic network of tubules, which encompass a contiguous lumen. The dynamics and unique shape of this organelle are regulated by the expression of several classes of ER-shaping proteins, many of which are conserved from yeast to humans. My preliminary results indicate that several of these proteins act as regulators of CVB and DENV infection. Thus, we hypothesize that enteroviruses and flaviviruses target specific ER-shaping proteins to facilitate their replication. The first set of proposed studies are designed to delineate the mechanisms by which reticulons, ER-shaping proteins that promote membrane curvature, regulate infection by CVB and DENV. Furthermore, we will determine the role of the insect reticulon homolog during DENV infection of insect cells. The second set of proposed studies will characterize the role of FAM134B during DENV infection. FAM134B is a recently identified ER-shaping protein that regulates autophagy-mediated degradation of the ER (ER-phagy). Results from these studies will provide details on novel host-pathogen interactions that mediate replication of two RNA viruses, which represent a significant public health burden. Information derived from our results will provide significant insight into the biology of these viruses that can be utilized in the development of antivirals to combat disease. Furthermore, our results will also help to define the specific role of ER-shaping proteins in regulating the shape and dynamics of the ER.